Leishmaniasis is a widespread and debilitating protozoal disease of man and animals. An improved understanding of the immunologic aspects of this infection should provide the basis for new therapies. We propose to study how IL-12 can be used to promote the development of a protective immune response, specifically focusing on the development of a therapeutic vaccine for chronic cutaneous leishmaniasis. Experimental infections in mice mimic to a large extent the spectrum of clinical presentations associated with human disease, and have been used extensively to study immunologic responses in leishmaniasis. From such studies we have learned a great deal about the factors involved in the differentiation and regulation of T cell subsets. Our hypothesis is that during chronic leishmanial infections mice are non-responsive to IL-12, leading to the inability to promote the development of a Th1-type immune response. We will define the factors that regulate IL-12 responsiveness, by identifying the cells that need to respond to IL-12 in order to shift a T cell population from a Th2 to a Th1 phenotype, defining the requirements for CD28-B7 interactions for effective IL-12 function, and determining what regulates the IL-12 receptor during Leishmania infection. Our approach will be to manipulate in vitro and in vivo levels of cytokines and costimulatory interactions to promote a shift in the Th2 population from L. major infected BALB/c mice towards a protective Th1 phenotype. We will use an in vitro system to define the cells and cytokines involved in switching, and we will then validate our in vitro findings with in vivo studies. Because antigen-reactive cells are difficult to track, due to their low frequency in a normal (or even infected) animal and the lack of reagents that specifically identify them, for some studies we will use cells from an OVA T cell receptor (TCR) transgenic mouse (DO11.10) in combination with an OVA expressing L. major parasite. The expression of OVA by L. major establishes this antigen as a model parasite antigen, that will be exposed to the same environment that shapes the T cell response to other antigens produced by L. major. An additional advantage to using DO11.10 TCR transgenic mice is that regulation of the IL-12 receptor by these T cells has been well studied. We will apply the information gained from these in vitro studies to design the best approach for promoting a Th1 response in infected L. major infected mice using a therapeutic vaccine.